Contrast agents and methods for preparing contrast agents

ABSTRACT

Contrast agents comprising a scaffold protein having at least one operative integrated metal ion binding site.

STATEMENT OF RELATED APPLICATIONS

This application claims priority on U.S. Provisional Patent ApplicationNo. 60,699,409 entitled “Contrast Agents and Methods for PreparingContrast Agents” having a filing date of 13 Jul. 2005, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to diagnostic imaging, and more particularly tonovel contrast agent preparations and their use in diagnostic imaging,for example in visualizing tissue.

2. Prior Art

Imaging technology, including magnetic resonance imaging (MRI), X-ray,positron emission tomography (PET), magnetography, and computedtomography (CT) scanning, has a vital role in the detection andtreatment of cancer lesions and other illnesses. For example, MRItechnology provides a powerful, non-invasive tool to map and explore thestructure and function of soft tissues. In fact, MRI through the use ofhigh-strength magnets and radio-frequency signals can producethree-dimensional images of tissues. Using mechanical imaging system, itis possible to detect neoplastic lesions; the detection of early tumorlesions and metastases still remains challenging.

Contrast agents have been used to improve the intrinsic contrast of theimages from imaging technology. This method relies on the administrationof contrast agents to amplify the contrast in imaging between thepathological tissue and the normal tissue. The most widely used class ofMRI contrast agents are based on gadolinium ion (Gd³⁺), manganese ion(Mn²⁺), and iron ion (Fe³⁺) chelates that are strictly extracellular lowmolecular weight compounds with T1 reflexivity such asdiethylenetriaminepentaacetate (DTPA). Ultimately, the efficacy of acontrast agent depends on both the inherent capability to improve imagesand the pharmacokinetics.

For example, the Gd³⁺ based contrast agents approved for clinical useare mainly non-specific small molecules. Such Gd³⁺ contrast agentsusually have relaxivities of <10 mM⁻¹ s⁻¹ which are 20 to 50 fold lowerthan the predicted values. The relaxivities are mainly limited by therotational correlation time of the molecule. The most commonly usedcontrast agent, DTPA, has a R1 relaxivity of 5 mM⁻¹ s⁻¹. With thisrelaxivity, a robust clinical examination usually requires a large dose(>0.1 mM local concentration) in order to reach sufficient contrast orto produce an acceptable image. In addition, this class of contrastagents has a very short circulation time that limits the time window fordata collection. Efforts to improve such contrast agents have includedthe covalent or the non-covalent linkage of the small Gd agent to themacromolecules, such as dendrimers or copolymers.

Accordingly, there is always a need for improved contrast agents withhigher capabilities to enhance imaging signals. There also is a need fornovel protein-based MRI contrast agents with wide applicability inmolecular imaging of various tissues, tumors, cancers, and diseases.There also a need for contrast agents that can be retained longer intissue and blood vessels, especially in that of animals and humans.There also is a need for contrast agents in which the chelating site maybe tailored for specific applications and the imaging techniques. It isto these needs among others that this invention is directed.

BRIEF SUMMARY OF THE INVENTION

Briefly, this invention is directed to a novel group of contrast agentshaving tuned properties for diagnostic imaging. More particularly, thisinvention is directed to a class of magnetic resonance imaging contrastagents that accumulates in tissue. The novel contrast agents comprise ascaffold protein that can be an organic polymer such a protein and atleast one tailored metal ion binding site capable of chelatingparamagnetic and heavy metal ions. The at least one tailored metal ionbinding site is integrated into select folding pockets within thescaffold protein. In most cases, more than one site can be integratedinto the scaffold protein.

The novel contrast agents can be developed by designing tailored bindingsites and integrating these sites into scaffold proteins. The bindingsite can be developed by a design approach or by a grafting approach.Further, other approaches, known or developed hereafter, can be used todesign binding sites suitable with this invention. After the site hasbeen developed, the site or sites are operatively integrated into theselect areas of the scaffold protein. The contrast agent then may beadministered to animals or humans through known delivery methods.

These features, and other features and advantages of the presentinvention, will become more apparent to those of ordinary skill in therelevant art when the following detailed description of the preferredembodiments is read in conjunction with the appended drawings in whichlike reference numerals represent like components throughout the severalviews.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary contrast agent prepared according to thisinvention.

FIG. 1B shows another exemplary contrast agent prepared according tothis invention.

FIG. 1C shows another exemplary contrast agent prepared according tothis invention.

FIG. 1D shows another exemplary contrast agent prepared according tothis invention.

FIG. 1E shows another exemplary contrast agent prepared according tothis invention.

FIG. 2 is schematic diagram of a contrast agent according to anillustrative embodiment of this invention.

FIG. 3A shows MR images produced by various contrast agents and DTPA.

FIG. 3B shows the proton relaxivity of different contrast agentscompared to DTPA.

FIG. 4A shows the dynamic properties of an engineered metal bindingprotein with discontinuous ligand residues.

FIG. 4B shows the dynamic properties (T1 time) of CA1-CD2 with acontinuous metal binding site.

FIG. 5A is a diagnostic image of a mouse prior to being administered anexemplary contrast agent according to this invention.

FIG. 5B is another diagnostic image of a mouse that has beenadministered an exemplary contrast agent according to this invention.

FIG. 5C is another diagnostic image of a mouse that has beenadministered an exemplary contrast agent according to this invention.

FIG. 5D is another diagnostic image of a mouse that has beenadministered an exemplary contrast agent according to this invention.

FIG. 6 shows the viability of various contrast agents according to thisinvention in human serum.

DEFINITIONS

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Forpurposes of the present invention, the following terms are defined.

The term “nucleic acid molecule” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single-strandedor double-stranded form, and, unless specifically indicated otherwise,encompasses polynucleotides containing known analogs of naturallyoccurring nucleotides that can function in a similar manner as naturallyoccurring nucleotides. For example, This term can refer to single anddouble stranded forms of DNA or RNA. Nucleic acid sequences are readilyapparent from

The term “recombinant nucleic acid molecule” refers to a non-naturallyoccurring polynucleotide containing two or more linked polynucleotidesequences. A recombinant nucleic acid molecule can be produced byrecombination methods, particularly genetic engineering techniques, orcan be produced by a chemical synthesis method. A recombinant nucleicacid molecule can encode a fusion protein, for example, a fluorescentprotein linked to a polypeptide of interest. The term “recombinant hostcell” refers to a cell that contains or can express a recombinantnucleic acid molecule.

The term “encoding” in the context of a polypeptide refers to thetranscription of the polynucleotide and translation of the mRNA producedtherefrom. The encoding polynucleotide is considered to include both thecoding strand, whose nucleotide sequence can be identical to an mRNA, aswell as its complementary strand. It will be recognized that encodingpolynucleotides are considered to include degenerate nucleotidesequences, which encode the same amino acid residues. Nucleotidesequences encoding a polypeptide can include polynucleotides containingintrons and exons. Nucleic acid sequences are readily apparent fromamino acid sequence and vice versa.

The term “control sequences” refer to polynucleotide sequences that arenecessary to effect the expression of coding and non-coding sequences.Such control sequences can include a promoter, a ribosomal binding site,and a transcription termination sequence. The term “control sequences”is intended to include, at a minimum, components whose presence caninfluence expression and can also include additional components whosepresence is advantageous. For example, leader sequences and fusionpartner sequences are control sequences.

The term “operatively incorporated” or the like refers to polypeptidesequences that are placed in a physical and functional relationship toeach other. In a most preferred embodiment, the functions of thepolypeptide components of the chimeric molecule are unchanged comparedto the functional activities of the parts in isolation. For example, afluorescent protein can be fused to a polypeptide of interest and in thefused state retain its fluorescence while the polypeptide of interestretains its original biological activity.

As used herein, the term “brightness,” with reference to a fluorescentprotein, is measured as the product of the extinction coefficient (EC)at a given wavelength and the fluorescence quantum yield (QY).

The term “probe” refers to a substance that specifically binds toanother substance (a “target”). Probes include, for example, antibodies,polynucleotides, receptors and their ligands, and generally can belabeled so as to provide a means to identify or isolate a molecule towhich the probe has specifically bound.

The term “polypeptide” or “protein” refers to a polymer of two or moreamino acid residues. “Polypeptides” or “proteins” are polymers of aminoacid residues that are connected through amide bonds. As defined herein,peptides are inclusive of both natural amino acids and unnatural aminoacids (e.g. beta-alanine, phenylglycine, and homoarginine). While aminoacids are alpha-amino acids, which can be either of the L-optical isomeror the D-optical isomer, the L-optical isomers are preferred. Such aminoacids can be commonly encountered amino acids that are not gene-encoded,although preferred amino acids are those that are encodable.

The term “isolated” or “purified” refers to a material that issubstantially or essentially free from components that normallyaccompany the material in its native state in nature. Purity generallycan be determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis, high performance liquidchromatography, and the like. A polynucleotide or a polypeptide isconsidered to be isolated when it is the least predominant speciespresent in a preparation.

The term “naturally-occurring” refers to a protein, nucleic acidmolecule, cell, or other material that occurs in nature. A naturallyoccurring material can be in its form as it exists in nature, and can bemodified by the hand of man such that, for example, it is in an isolatedform.

Two or more amino acid sequences or two or more nucleotide sequences areconsidered to be “substantially identical” or “substantially similar” ifthe amino acid sequences or the nucleotide sequences share at least 80%sequence identity with each other, or with a reference sequence over agiven comparison window. Thus, substantially similar sequences includethose having, for example, at least 85% sequence identity, at least 90%sequence identity, at least 95% sequence identity, or at least 99%sequence identity.

Two or more amino acid sequences or two or more nucleotide sequences areconsidered to be “similar” if the amino acid sequences or the nucleotidesequences share at least 50% sequence identity with each other, or witha reference sequence over a given comparison window. Thus, substantiallysimilar sequences include nucleotide sequences considered to be“substantially identical” or “substantially similar”.

The term “fluorescent properties” refers to the molar extinctioncoefficient at an appropriate excitation wavelength, the fluorescencequantum efficiency, the shape of the excitation spectrum or emissionspectrum, the excitation wavelength maximum and emission wavelengthmaximum, the ratio of excitation amplitudes at two differentwavelengths, the ratio of emission amplitudes at two differentwavelengths, the excited state lifetime, or the fluorescence anisotropy.

The term “fluorescent protein” refers to any protein capable of lightemission when excited with an appropriate electromagnetic energy.Fluorescent proteins include proteins having amino acid sequences thatare either natural or engineered, such as the fluorescent proteinsderived from Aequorea Victoria fluorescent proteins.

The term “mutant” or “variant” also is used herein in reference to afluorescent protein that contains a mutation with respect to acorresponding wild type fluorescent protein. In addition, reference ismade herein to a “spectral variant” or “spectral mutant” of afluorescent protein to indicate a mutant fluorescent protein that has adifferent fluorescence characteristic with respect to the correspondingwild type fluorescent protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments of this invention include contrast agents capable ofenhancing image contrast by affecting water molecule proton relaxationrates. Such contrasts agents are effective for magnetic resonanceimaging, in part, because the water proton relaxation rate in the targettissue is affected differently than the relaxation rate of the waterprotons in the surrounding tissue. The contrasts agents as disclosedherein are paramagnetic species, which form complexes with metal ions,so to alter the relaxation rates of adjacent nuclei.

More particularly, this invention is directed to a novel group ofdiagnostic contrast agents having tuned properties, even moreparticularly, to a class of magnetic resonance contrast agents thataccumulates in tissue. The novel contrast agent comprises (a) a scaffoldprotein that can be an organic polymer such a protein and (b) at leastone tailored metal ion binding site capable of chelating paramagneticand heavy metal ions, wherein the at least one tailored metal ionbinding site is integrated into select folding pockets within thescaffold protein.

The novel contrast agents can be developed by designing tailored bindingsites and operatively integrating these sites into scaffold proteins. Aswill be discussed later in more detail, the binding site may bedeveloped by a design approach or by a grafting approach. After the sitehas been developed, the site or sites are operatively integrated intothe select areas of the scaffold protein. The contrast agent then may beadministered to animals or humans through known delivery methods.

In illustrative embodiments, at least one of the metal chelating sitesis embedded in the scaffold protein. In such an embodiment, the metalchelating site can be placed within the scaffold protein such that themetal chelating sites are within the interior of the contrast agent.Preferably, at least one of the metal chelating sites is embedded usingamino acids of the scaffold proteins as ligands to chelate the metalion. More preferably, the at least one metal binding site is embeddedwithin the protein such that the scaffold protein has a correlation atleast in part resembling the protein itself.

In illustrative embodiments, the scaffold protein for MRI applicationsis a protein that will host the tailored metal ion binding sites and hasthe following characteristics:

(a) stability in a physiological environment against cleavage anddenaturation;

(b) a topology suitable for the integration of metal ion sites;

(c) a rotational correlation time optimized for the magnetic field (e.g.around 100 milliseconds in a magnetic flied of 1.3 to 3T), e.g. highermagnetic field application can prepared by changing the site of theprotein; and

(d) a water exchange rate such that the relaxivity of the protein is notlimited by the water exchange rate.

Preferred properties of the scaffold protein also may include watersolubility, low interaction with the other cellular metal ions and lowtoxicity. While of all these properties are not required, the optimalproperties of the scaffold protein can and do depend on the specificparameters of the imaging application.

One important property of the scaffold protein is its ability to acceptthe introduction of metal ion binding sites therein. Preferably, thescaffold protein has a folded conformation, three-dimensional structureor an amino sequence with some homology to the proteins whose structurehas been solved at least in part. For example, the scaffold protein canbe screened to determine whether it can tolerate the integration ofvarious binding sites without excessive denaturation. For example, theintegration of metal ion binding sites into the scaffold protein shouldnot denature or unfold the protein. Thus, the metal ion binding siteshould not be placed by mutating a hydrophobic core or in a positionthat results in substantial structural perturbation. This can beexamined by sequence alignment of proteins in the same family.Preferably, the amino acids that have an essential role in folding ofthe structure or the function will be conserved among different speciesof this same type of the protein.

In another embodiment, the scaffold protein can be a natural proteinthat chelates a metal ion. In such embodiments, it is possible to modifythe natural metal binding sites to chelate heavy metals or paramagneticmetals or other metals useful in diagnostic imaging. For example, it ispossible to tailor the amino acid sequence of the scaffold protein thatordinarily binds Ca²⁺ to bind Gd³⁺ by modifying nitrogen or oxygenmolecules contained therein.

Preferably, metal ion binding sites are placed into a scaffold proteinsuch that the metal is able to be tumbled together with the protein. Itis better to find a location that is not as flexible as or is the sameflexibility as the protein body so as to match the correction time. Inthis case, it is preferred to design or create the binding pocket in theprotein. Although insertion also should work, it is preferable to do soin a relatively not so flexible region. Usually the protein can bechecked by looking at the B factor (temperature factor for X-ray) or S²factor (dynamic flexibility factor for NMR) of the pdb (protein databank) file of the structure.

More than one metal binding site may be integrated into a scaffoldprotein. The inclusion of more than one binding site improves thesensitivity of the contrast agent. Further, in cases where more than onebinding site is integrated into the protein, the site could havedifferent affinities but should still have strong enough affinity forthe selected metal so to avoid competition with physiological metalions. Both metal ions should be embedded into the host protein withpreferred rotational correlation times and water exchange rates toprovide MRI contrast with an increased sensitivity.

In preferred embodiments, the contrast agents can have a high affinityto and can preferentially select a particular metal ion (e.g. Gd³⁺, Mn²⁺or Fe³⁺). In one example, exemplary contrast agents showed adissociation constant K_(d) less than 10⁸ [M] for Gd³⁺ in an environmenthaving physiological metal ions and prevented those metal ions fromprecipitation under physiological conditions. Thus, the presentinvention may be used to create contrast agents having optimalselectivity for a specific metal ion.

The present invention can provide a new mechanism to increase therelaxivity of contrast agents. This is accomplished by designing themetal ion binding sites, e.g. Gd³⁺, in proteins, which can eliminate themobility and flexibility of the chelating moiety associated withcurrently available contrast agents. More particularly, by tailoring thebinding site, it is possible to prepare contrast agents with higherrelaxivity. High proton relaxivity by contrast agents can furtherenhance images.

One advantage of the present invention is that it provides contrastagents that can preferentially chelate a specific metal ion. Forexample, a preferred contrast agent having Gd³⁺ binding site(s) willpreferentially chelate Gd³⁺ over other metal ions, such Mg²⁺ or Ca²⁺. Asheavy metals tend to be toxic to cells and animals, contrast agents thatare able to form a stronger bond with the metal ion are less toxic. Assuch, the ability to preferentially chelate a specific metal ion canimprove the specificity of a contrast agent and can reduce thecytotoxicity of the contrast agent.

In another embodiment, a fusion protein or a non-degradable particlemoiety can be added to the protein contrast agent with a linker to tunethe correlation time for optimal contrast sensitivity, targeting(subcellular, cellular, tissue and organ selectivity), biodistribution,and bioelimination. One of ordinary skill in the art may determine suchlinkers without undue experimentation.

In another embodiment, this invention can be used to producemicrospheres of contrast agents of low density. These microspheres canhave an internal void volume that can be at least about 75% of the totalvolume of the microsphere. The microspheres may be of varying size,provided they are low density. Suitable size microspheres include thoseranging from between about 1 and about 1000 microns in outside diameter.

The contrast agents of the present invention may be formulated withconventional pharmaceutical or veterinary mechanisms and materials. Thecontrast agent compositions of the present invention may be inconventional pharmaceutical administration forms such as powders,solutions, suspensions, dispersions, etc.; however, solutions,suspensions and dispersions in physiologically acceptable carrier media,for example water for injections, will generally be preferred. Forexample, such materials include emulsifiers, fatty acid esters, gellingagents, stabilizers, antioxidants, osmolality adjusting agents, buffers,preservatives, antimicrobial agents, and pH adjusting agents. Thecompositions according to the invention can therefore be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art.

The administration of the contrast agents of this invention can be donein a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. Preferably, the delivery mechanisms include parenteraladministration (injection or infusion directly). Depending upon themanner of introduction, the compounds may be formulated in a variety ofways. The concentration of active compound in the formulation may varyfrom about 0.01-100 wt. %.

One of the bioelimination routes for the contrast agents of thisinvention can be renal. The macrostructure is eventually to beabstracted by the RES and it is preferred that chelate attachment shouldbe via biodegradable bonds that on cleavage release fragments that arerenally excretable, e.g. with a molecular weight of less than 60 KDa,preferably less than 10 KDa, especially 200 to 5000 Da. To alter thebio-elimination route, a fusion protein or a non-degradable particlemoiety can be added to the protein contrast agent.

To overcome immunogenicity, the contrast agent can be modified for usewith the specific organism by those with ordinary skill in the art. Forexample, where the contrast agent in used in rats, the contrast agentmay be modified by incorporating the rat self sequence. Further, it iscontemplated that the contrast agent could include human sequences. Anadditional advantage of the protein-based agent of the present inventionis that it is relatively easy to target the specific tissues andbiomarkers for molecular imaging of tissues and tissue growths such ascancer. The active targeting of contrast agents to specific organs ortissues can be achieved by incorporation of lipids with monoclonalantibodies or antibody fragments that are specific for tumor associatedantigens, lectins or peptides attached thereto.

Scaffold Proteins.

Scaffold proteins suitable with the present invention include proteinsor organic polymers containing amino acids. Such scaffold proteins areinclusive of both natural amino acids and unnatural amino acids (e.g.beta-alanine, phenylglycine, and homoarginine). While amino acids arealpha-amino acids, which can be either of the L-optical isomer or theD-optical isomer, the D-optical isomers may be preferred, as suchisomers are less subject to proteolytic degradation. Such amino acidscan be commonly encountered amino acids that are not gene-encoded,although preferred amino acids are those that are encodable.

Various scaffold proteins may be used according to the invention but ingeneral they will be proteins, terminally modified proteins, and organicpolymers. More specifically, suitable scaffold proteins can be selectedwith properties suitable for diagnostic applications. The scaffoldprotein for use with this invention may be of unitary construction (aparticulate, a polychelant or a dendrimeric polymer). Scaffold proteinssuitable with this invention may be selected without undueexperimentation.

The scaffold protein also can be a natural protein that ordinarily bindsa metal ion. In such embodiments, it is possible to modify the naturalmetal binding sites to chelate heavy metals or paramagnetic metals orother metals useful in diagnostic imaging. For example, it is possibleto tailor the amino acid sequence of the scaffold protein thatordinarily binds Ca²⁺ to bind Gd³⁺ by modifying amino acid ligandresidues contained therein. For example, one can modify the bindingsites in alpha-lactalbumin to bind Gd³⁺ (e.g. as shown in FIG. 3). Foranother example, it is possible to modify EF-hand calcium binding sitesin proteins such as calmodulin to bind Gd³⁺ (e.g. CA9.CD2).

In illustrative embodiments, a scaffold protein can be selected for thefollowing criteria:

1) Exhibition of strong stability in terms of resistance to pHdenaturation and resistance to proteolytic cleavage.

2) The availability of structural information about the protein. If lessstructural information is available, which allows for the rationaldesign of metal binding sites with optimized inner, secondary and outersphere relaxation and metal binding properties, then structureprediction can allow for the modification of the protein.

3) Tolerance of mutations without sacrificing native conformation andfolding.

4) The molecular sizes are suitable for the particular application. Anoptimal size can be dependant on a particular diagnostic application.For example, a compact structure, e.g. molecular weights between 11-30KDa, and rotational correlation times of ˜10-30 ns. Further, a molecularsize can improve circulation retention times and tissue penetration. Forexample, stronger in vivo kidney images and prolonged retention time canallow for more detailed imaging of the renal system for diagnosingkidney diseases such as renal carcinoma and can allow for more precisemeasurement of blood flow and volume. Further, a proper size of theprotein frame can provide improved tissue penetration and moleculartargeting, which can be a limitation of some the large size ofdentrimers, nano-particles, and superparamagnetic particles.

5) Optionally, the scaffold protein also can have intrinsic properties,which can allow for the construction of multifunctional probes and useof fluorescence as a tool to assist in the design of MRI contrast agentsfor molecular imaging without the need of other fluorophores.

Suitable proteins include proteins from immunoglobulin G (IgG)superfamily such as CD2 proteins (a cell adhesion protein) that exhibithigh stability against proteolysis, thermal conditions (Tm 67 C), pH(2-10), and salt (0-4 M NaCl) denaturation. CD2 proteins can be suitablewith this invention because such proteins are stable in physiologicalenvironments, have a topology suitable for the integration of at leastone or multiple metal ion chelating sites, and typically have arelaxivity greater than 10 mM⁻¹ s⁻¹ (some of them up to about 50 mM⁻¹s⁻¹). In addition, CD2 can tolerate multiple surface mutations withoutunfolding the protein. Other research has shown that CD2 can be used asa host protein to design calcium binding sites. Examples using CD2 aredescribed below.

Fluorescent proteins are another class of preferred scaffold protein forthis invention, as these proteins are stable in a physiologicalenvironment against proteolytic degradation and pH denaturation (pH5-10). Such fluorescent proteins include an array of fluorescentproteins including those related to Aequorea. Suitable fluorescentproteins should have a useful excitation and emission spectra and mayhave been engineered from naturally occurring Aequorea victoria greenfluorescent proteins (GFPs). Such modified GFPs may have modifiednucleic acid and protein sequences and may include elements from otherproteins. The cDNA of GFPs may be concatenated with those encoding manyother proteins—the resulting chimerics are often fluorescent and retainthe biochemical features of the partner proteins. Yellow fluorescentproteins and blue fluorescence proteins and red fluorescent proteins canalso be used to as the scaffold proteins for contrast agents. Suchproteins also are included in the invention.

Other suitable proteins include extra cellular receptors and growthfactors are known to be stable against protein cleavage. In addition,proteins from four-helical bundle family (such as Rop), the maltosebinding protein family, and thioredoxin family have been shown to acceptmutations and metal binding sites. While the inventors have not testedevery protein for suitability as a scaffold protein, the diverse arrayof examined proteins demonstrates this invention includes all of theproteins having the criteria disclosed herein. It is contemplated thatone of ordinary skill in the art can develop and select a suitablescaffold protein based using ordinary research techniques and thecriteria disclosed herein.

One advantage of using fluorescent proteins is that contrast agentsconstructed from such proteins can be multi-functional probes. In suchan embodiment, the contrast agent constructed from fluorescent proteinscan be screened using both fluorescence and MR imaging. This can beextremely advantageous as such properties equip the contrast agent withboth the fluorescence needed for fluorescence detection methods and thesensitivity needed for the deep tissue detection from MRI. Such contrastagents are multifunctional contrast agents.

Other proteins may be used as scaffold proteins for this invention.Preferably, scaffold proteins are able to tolerate the addition of themetal ion binding site without substantial disruption to its structure.One of ordinary skill in the art can select a scaffold protein based onpreferences without undue experimentation.

Metal Ion Binding Sites

The affinity of the metal ion binding site may vary the contrast agentaffinity for metal ions. Specifically, as affinity and sensitivity ofthe metal ion binding sites may be modified, the relaxivity and metalaffinity of the contrast agent may be modified. Preferably, the metalion binding site has optimal imaging properties including metal bindingaffinity, selectivity, relaxivity, nuclear magnetic relaxationDispersion (NMRD) profile, and water exchange rates.

One of ordinary skill in the art can uses methods known in the art ordeveloped hereafter to develop a metal binding site having optimalcharacteristics. For example, the metal ion binding site of the presentinvention can be constructed at least using these methods:

(1) A computational design approach in which the metal ion binding sitewith a selectivity and affinity for a metal ion is engineered andrationally designed de novo based on optimal binding characteristics ofmetal ion with other moieties;

(2) A grafting method in which the metal ion binding site with aselectivity and affinity for a metal ion is engineered and constructedselectively by varying the primary, secondary, tertiary, and/orquaternary structures of an identified binding site; and

(3) Other methods known or developed hereafter and a combination ofmethods known or developed hereafter.

1. The Computational Design Approach

The computational design approach focuses on designing a metal ionbinding site de novo. This design approach focuses on using an algorithmto construct and engineer an optimal binding site. Preferably, thecomputation design approach is used to create optimal binding sites by,e.g., varying the coordination geometry of the site, the water number inthe coordination shells, the ligand types, and the charges.

The computational design approach comprises the following steps:

(1) Accessing one or more databases having structural, coordination,and/or 3-dimensional structures or models of metal ion binding sites, orcreating model structures based on the sequence homology to other metalbinding sites;

(2) Generating one or more preliminary metal ion binding sites fromportions of the structural data;

(3) Selecting rationally one or more suitable metal ion binding sitesfrom the generated preliminary binding sites based on, e.g.,coordination geometry; and

(4) Creating a metal ion binding site by tailoring and tuning theselected metal ion binding site.

The metal ion binding site may be incorporated into a scaffold protein,e.g. a fluorescent or CD2 protein. Further, such a method may be used toalter metal ion binding properties of proteins and generate newmaterials with various ion binding affinities.

More particularly, the method involves searching and accessing publicand or private databases for preferred components of a metal ion bindingsite. Such databases that may be searched for the criteria or componentsmay include public domain banks (e.g. National Center for BiotechnologyInformation (NBCI) or PubMed of the US National Institution of Health)or knowledge banks such as protein modeling structure data banks (e.g.Cambridge or RCSB Protein Data Bank Data Bank and BioMagResBankdatabase) or other biotechnological data banks. Further, the databasecould include structural data from metal ion binding proteins whosestructures have been characterized previously. One of ordinary skill inthe art can identify databases and sources of material for databasessuitable with this invention. Use of a computer with internet orintranet capabilities obviously would greatly speed up the searching andis preferred.

These databases may be used to provide structural analysis of one toseveral thousand different small molecules or metal ions that bind to aprotein. Such analysis may include local coordination properties, typesof residues or atoms commonly used to bind a desired metal ion, chemicalfeatures (e.g. pKa or changes), the number of charged residues on asite, and the range or deviation of the known binding sites. Further,such analysis may include the environment, such as types of atoms,residues, hydrophobicity, solvent accessibility, shapes of the metalbinding sites, electrostatic potentials, and the dynamic properties(e.g. B-factors or the order factors of the proteins) of the bindingsites. Such analysis also may include whether binding site for aparticular metal ion is a continuous or discontinuous binding site.

Once preliminary metal ion binding sites are found, using the structuraldata and analysis, one or more suitable metal ion binding sites may begenerated based on rational factors. Specifically, different searchalgorithms may be used to generate potential metal ion binding sitesbased on other key features in addition to, for example, the geometricdescriptors. These key features include the properties of the originalresidues in the scaffold protein, ligand positions that are essential toprotein folding, the number of the charged residues and theirarrangement and number of water molecules in the coordination shell. Thehydrogen bond network and the electrostatic interactions with thedesigned ligand residues also can be evaluated. Furthermore, the proteinenvironments of metal ion binding sites can be analyzed according tosolvent accessibility, charge distribution, backbone flexibility, andproperties of scaffold proteins. Thus, one of ordinary skill in the artmay rationally select a binding site based on desired parameters.

Once the metal ion binding sites are generated, a site may be tailoredusing two complementary approaches of computational design and grafting(see below). First, as discussed above, the metal ion binding site maybe tailored using a grafting method in which the primary, secondary,tertiary, and/or quaternary structures are tuned. Second, the metal ionbinding site may be tailored using a computational design approach. Itis understood that one or both of these approaches may be used to tailorthe binding site.

The computational design approach includes modifying the metal ionbinding site by modifying residues in the scaffold of the metal ionbinding site. In one embodiment, a geometric or statistical descriptionof the ligands around a metal ion, a three-dimensional structure of thebackbone of proteins, and a library of side-chain rotamers of aminoacids (or atoms from the main chain) can identify a set of potentialmetal-binding sites using a computer. Using the geometric and graphdescription of a particular metal ion site, key ligand residues arecarefully placed in the amino acid sequence to form the metal (metalion) binding pocket. This binding pocket can be created automatically bya computer algorithm designed according to the coordination descriptionand the user's preferred affinity.

The created potential metal ion binding sites can be optimized and tunedto specification. A backbone structure of the metal ion binding sitewith different degrees of flexibility may be used according to the needor the flexibility of the metal ion binding site. The designed metal ionbinding sites are further filtered and scored based on the localfactors, which may include the shape of the metal ion binding sites,locations, charge numbers, dynamic properties, the number of mutationsneeded, solvent accessibility, and side chain clashes. To achieve themaximum relaxivity, it can be important to have one to two oxygen atomsfrom the solvent water molecules in the coordination shell withoutreducing the required binding affinity and selectivity.

Stronger metal ion binding affinities of the designed sites may bedeveloped based on several modeled factors that contribute to metal ionaffinity. For example, the number of ligand residues is a factor todirectly chelate a specific metal ion. In some cases, in order to have astrong metal ion affinity with a K_(d) necessary to measure a metal ionconcentration, it is necessary to include residues from the proteinframe for optimal metal ion binding. In other cases, the number ofcharged residues is able to change metal ion affinity. In still othercases, the ligand type is a factor as the binding preferences of achelate may depend on the particular ligand type. Other factors, such asnegatively charged environments, may contribute to the binding affinityof a metal ion binding protein and can be taken into account by those ofordinary skill in the art without undue experimentation. These chargedresidues can increase the water-exchange rate to avoid its limitationfor the required relaxivity.

An illustrative version of this computational approach is thecomputerized (or otherwise automated) querying of one or more databasesthat comprise structural data on metal ion binding sites using selectedcriteria relevant to the metal ion binding site, generating at least onepreliminary metal ion binding site from the database information basedon compatibility with the selected criteria, and selecting one or moresuitable metal ion binding sites from the preliminary metal ion bindingsites based on optimal compatibility with the selected criteria. Once asuitable metal ion binding site is selected, the nucleic acid sequenceof the selected metal ion binding site is obtained, tailored, andoperatively linked with a scaffold protein sequence, whereby the nucleicacid sequence of the selected metal ion binding site is tailored so toachieve the metal ion binding site having a desired specificity for themetal ion. Further, a nucleic acid sequence encoding the preliminarybinding sites can be generated from the structural or model data. Thecomputational approach also can be used to produce the metal ion bindingsite.

The computational approach can be performed on or by a system comprisingat least one database that comprises the structural data on metal ionbinding sites, an algorithm for generating the preliminary metal ionbinding sites from portions of the structural or model data usingselected criteria relevant to the metal ion binding site and rating thepreliminary metal ion binding sites based on specificity for a selectedmetal ion, and a computer for executing the algorithm so as to query thedatabases to generate the preliminary metal ion binding sites. Thealgorithm generally is a relatively simple searching algorithm that willquery the databases based on inputted criteria.

2. The Grafting Method

The grafting method focuses on engineering and constructing a metal ionbinding site by modifying the primary, secondary, tertiary, and/orquaternary structure of an identified binding site. By selectivelymanipulating the structure of the binding site, it is possible to obtaina metal ion binding site that can be engineered into a scaffold protein,e.g. CD2 or fluorescent protein, without significantly denaturing theprotein. Using the grafting method, it is possible to achieve a bindingsite that has a stronger preference for one metal ion over another metalion. Such modifications may allow for improved contrast abilities.

Initially, an identified binding site for use with the grafting methodmay be any continuous sequence site that has some affinity for a metalion. Such binding sites may derive from either known binding peptidessuch as an individual EF-hand site or from short fragments that havedemonstrated the ability to bind specific metal ions such asalpha-lactalbumin. Such peptides may be highly conserved in nature andprevalent throughout nature or may be unnatural but known to have anaffinity for a particular metal ion. One of ordinary skill in the art isable to identify binding sites with affinity for a metal ion withoutundue experimentation. Once the binding site has been identified, theprimary structure of the metal ion binding site may be altered and tunedto achieve a metal ion binding site with improved bindingcharacteristics. For example, more charged ligand residues suchaspartate and glutamate may be engineered by inserting codon(s) into themetal ion binding site so as to tune the responsiveness of the site orthe scaffold protein. The inclusion of additional charged ligands canallow the contrast agent to achieve an affinity for selectedparamagnetic metal ions and to have a desired selectivity. Additionally,one or two water molecules can also be introduced into the coordinationshell by removing or modifying ligand residues and their environments.Further, other mutations to the primary structure include removing oradding amino acids to change properties such as flexibility or rigidityof the site. Adding or removing amino acids from the binding site altersthe primary structure of the binding site.

The secondary structure of the metal ion binding site, that is, thespatial arrangement of amino acids residues that are near one another inlinear sequence, may be modified to tune the sensitivity andresponsiveness of the metal ion binding site. The residues on the siteitself, the flanking or the neighboring structures such as helices, betastrands, or turns may be modified by changing properties such ashydrophobicity, salt bridges, secondary structure propensity (e.g.helicity, and β-sheets), and charge interactions with different aminoacids, which all may inherently change the secondary structure.

The tertiary structure of the metal ion binding site may be modified tofurther tune the sensitivity and responsiveness of the metal ion bindingsite. The affinity of the metal ion binding site for the metal ion maybe varied by selectively manipulating and adding helices, loops, bridgesand/or linkers and chemical properties such as hydrogen bonding,electrostatic interactions and hydrophobic interactions. In fact, suchvariations in tertiary structure may add stability and affinity byincreasing secondary structure propensity, adding charge interaction ofthe side chains, and by stabilizing the metal ion binding coordinationchemistry. As such, it may be possible to increase or decrease thebinding affinity of the continuous binding site by tuning the tertiarystructure of the metal ion binding site. In addition, the dynamicproperties can be modified by increasing the packing of the protein andreplacing residues with amino acids or other moieties with more rigid(e.g. Pro) or flexible (e.g. Gly) properties, or adding disulfide bonds,

One method of directly altering the primary, secondary, and/or tertiarystructure of the metal ion binding site is by altering the charges inthe site. As the charges in any binding site have a significant role inthe structure of the site, changing the charges or charge ratio may havesignificant impact on the structure of the site. More importantly, asthe charged side chains exhibit a strong influence on the metal ionbinding affinity even though they are not directly involved as ligands,the variation of these chains results in variations in metal ion bindingaffinities and selectivity. A metal ion binding site may have strongeraffinities to and better selectivity for a desired metal ion over acompetitive metal ion by designing or modifying the site, e.g., changingthe number of charged ligand residues to form metal ion binding pockets.For example, the metal ion binding affinity of the metal ion bindingsite may be varied by changing the charged side chains that are presenton the metal ion binding site and or the neighboring environment. Thereplacement of charged residues such as aspartate or glutamate with aresidue such as alanine may dramatically reduce the binding affinity forthe metal ion by up to 100 times.

In the case of multifunctional contrast agents, e.g. where the contrastagent is a fluorescent protein, it can be important to induce metalbinding sites without altering significantly the chromophore environmentto reduce the fluorescent/optical signal. These metal binding sites canbe added at remote locations away from the chromophore or simple fusionto the fluorescent moieties. Such locations can be evident from thesequence and protein folding.

In other embodiments, the grafting approach may be used with the designapproach to create optimal metal binding sites. For example, metalbinding sites can be created by using part of a continuous binding siteand part of ligand residues created by computer design. The loops or anysequences of the proteins can be removed or modified to achieve optimalrequired binding affinity, metal selectivity, relaxivity and stability.Thus, by varying the primary, secondary, and/or tertiary structure ofthe metal ion binding site, it is possible to achieve a metal ionbinding site with desired specificity and affinity and more importantlycontrast abilities.

3. Other Methods

The metal ion chelating or binding can be developed using methods knownor developed hereafter. Such methods include protein engineering methodsthat are readily available in the art, which include by modifying theexisting metal binding sites to change the metal binding specificity anddynamic properties. Such methods also include techniques to modifyexisting binding sites with protein ligand residues or fuse toprotein-contrast agents with other molecules, which include theformation of metal binding sites with other molecules/prosthetic groupsincluding non-natural amino acids or carbohydrates or phosphates.Exemplary methods for protein engineering or for design suitable methodsare also disclosed in Barondeau D. P. and Getzoff E. D., StructuralInsights into Protein-Metal Ion Partnerships, Current Opinion inChemical Biology, 2004, 14:7; and Lu, Y, Design and Engineering ofMetalloproteins Containing Unnatural Amino Acids or Non-NativeMetal-Containing Cofactors, Current Opinion in Chemical Biology, 2005,both of which are incorporated by reference in their entirety.

Further, it is possible to combine methods to prepare desired metal ionchelating sites.

Selecting Metal Ion Binding Sites in the Scaffold Protein

The metal ion binding sites may be selectively introduced into numeroussites of a scaffold protein without substantially impairing itssecondary structure. A number of methods for identifying integrationsites in proteins, such CD2 proteins, fluorescent proteins (e.g. GFP,YFP, CFP, and RFP) are known in the art, including, for example, sitedirected mutagenesis, insertional mutagenesis, and deletionalmutagenesis. Other methods, including the one exemplified below and inthe Examples, are known or easily ascertained by one skilled in art.

The sites of the fluorescent protein that can tolerate the insertion ofa metal ion binding site also may be determined and identified by genemanipulation and screening. By generating mutant proteins and bymanipulating the DNA sequence, it is possible to obtain a variety ofdifferent insertions, which then may be screened to determine whetherthe protein maintains its intrinsic activities. Preferably, sites thatremove or interfere with the intrinsic fluorescence of the fluorescentprotein are not optimal and may be screened out. Variants identified inthis fashion reveal sites that can tolerate insertions while retainingfluorescence.

The preferred metal ion binding sites for use with scaffold proteins maybe selected by considering five criteria so to as optimize the localproperties of the metal binding site, the fluorescent protein, and theprotein environment. First, the geometry of the metal ion binding siteshould have relatively minor deviations from the desired coordinationgeometry. Second, negatively charged residues should be varied by nomore than 3-5 charges according to the desired affinity for metal ion(K_(d)). Third, the water coordination shell of the metal ion chelatingsites should be able to coordinate at least 1-2 water molecules. Fourth,the residues from the loops between the secondary structures with goodsolvent accessibility are desired for both the folding of the proteinand the fast kinetics required for the contrast agent.

The mutation or the introduction of the metal ion binding site shouldnot substantially interfere with the synthesis and folding of theprotein. More particularly, the introduction of the metal ion bindingsite should not interfere with either post-translational chromophoreformation or intermolecular interactions required for stabilizing thechromophores and folding of the protein frame. Furthermore, theintroduced side chain should not be overpacked and should not clash withthe protein frame of the scaffold protein (e.g. the fluorescentprotein). The direct use of chromophore residues as chelating sites isnot preferred but is within the scope of this invention.

Metal Ions

Metal ions are atoms and ions, including the respective isotopes andradioisotopes, that can bind to proteins or peptides. A metal ion maybind reversibly or irreversibly and such a bond may be covalent ornon-covalent. While Gd³⁺ is used in preferred embodiments of thisinvention as an exemplary metal ion for MRI contrast agents, it isunderstood that metal ions suitable with this invention include, but arenot limited to metal ions including paramagnetic metal ions, transitionmetal ions, and Lanthanide Series ions. Exemplary metal ions include,but are not limited to, the ion, isotope, and/or radioisotope forms ofmagnesium, calcium, scandium, titanium, manganese, iron, boron,chromium, cobalt, nickel, cooper, zinc, gallium, strontium, yttrium,strontium, technetium, ruthenium, indium, hafnium, tungsten, rhenium,osmium, and bismuth. It is also possible to use radioisotopes of metalswith this invention. Paramagnetic metal ions are the preferred metalions for use with this invention.

The metal ions chosen to be chelated by the contrast agents depend inpart on the diagnostic role of the ion. Metals that can be incorporated,e.g. through chelation, include lanthanides and other metal ions,including isotopes and radioisotopes thereof. For MR imagingapplications, the preferred metal ion is paramagnetic metal ion such asgadolinium. One of ordinary skill in the art can select a metal ion forchelation, based on the intended diagnostic application, without undueexperimentation.

As mentioned, the choice of metal ions to be held in chelate complexesby the contrast agents of the invention depends upon the diagnostictechnique for which the agent is to be used. For MRI or MRS or EPRapplications, the metal ions should be paramagnetic (metal ions withunpaired electrons), and preferably non-radioactive. For X-ray andultrasound imaging, heavy metal ions, e.g. with atomic numbers of atleast 37, preferably at least 50, should be used, again preferablynon-radioactive species. For scintigraphy the metal ions should be ionsof radioactive isotopes. For MR, X-ray, EIT or magnetometric imaging,one may use chelating groups to bind to heavy metal clusters (e.g.polyoxoanions and full or partial sulfur analogues) or to iron oxides orother superparamagnetic polyatomic species.

Methods of complexing metal ions with chelants and polychelants areknown to those with ordinary skill in the art. Metal may be incorporatedinto contrast agent, i.e. the tailored binding sites, by directincorporation, template synthesis, and transmetallation. Preferably, themetal ion is chelated into the contrast by direct incorporation, whichinvolves titration with solution of sub-stoichiometric levels up to fullincorporation.

EXAMPLES

In following examples, the inventors have focused on disparate proteins,namely, immunoglobulin super-family proteins and fluorescent proteins.For example, domain 1 of the cell adhesion protein CD2 and the greenfluorescent protein were selected as scaffold proteins (e.g. FIGS. 1Aand 1B) for the integration of engineered various metal (e.g. Gd³⁺)binding sites for several reasons. Further, the 3D model structures of6D79 of CD2 variant with designed metal binding sites (ball) and themodel structures of 6D79 and 7E15 based on doamin1 of rat CD2 (1 hng).3D structure of GFP (1b9c) with designed Gd³⁺ sites and the chromophorehighlighted. The Gd³⁺-binding residues and the adjacent residues areshown. The residues close to strand B also are represented in thefigure. For example, FIG. 1A shows a 3D structure of CD2 with designedmetal binding site. The loops that undergo relatively large dynamicproperty changes are shown in orange. Only one residue is labeled whencontinuous residues are colored.

First, these proteins exhibit strong stability in terms of resistance topH denaturation and resistance to proteolytic cleavage. For example, theprior art has shown that GFP is extremely stable and cannot be cleavedby proteases such as trypsin, chymotrypsin and thrombin. Further,pervious research has shown that domain 1 of CD2 maintains its nativeconformation between pH 2 to 11.

Second, various structural information about CD2 and GFP is available,which allows for the rational design of metal binding sites withoptimized inner, secondary and outer sphere relaxation and metal bindingproperties.

Third, these proteins tolerate mutations without sacrificing theirnative conformation and folding.

Fourth, these proteins have a compact structure, molecular weightsbetween 11-30 KDa, and rotational correlation times of ˜10-30 ns. Theseproteins have been matched to the optimal relaxivity for the currentclinically allowed magnetic field strength.

Fifth, the molecular sizes are suitable for good circulation and tissuepenetration.

Sixth, in the case of GFP, the intrinsic fluorescence allows for theconstruction of multifunctional probes and the use of fluorescence as atool to assist in the design of MRI contrast agents for molecularimaging without the need of other fluorophores.

FIG. 2 shows an example of this methodology in which several Gd³⁺binding sites were designed at different locations of CD2. Gd³⁺, Tb³⁺,La³⁺ and the other lanthanide metal ions have coordination propertiessimilar to those of Ca²⁺. In the schematic representation of a Gd³⁺chelate with one inner-sphere water molecules surrounded by bulk watermolecule, τ_(M) refers to the rotational correlation time of themolecule, k_(ex) to the water/proton exchange rate or the reciprocal ofresidence lifetime of the coordinated water τ_(M) and 1/T_(1,2e) to therelaxation rate of Gd³⁺ electron spin. All have a strong preference foroxygen atoms from carboxyl sidechains of Asp, Glu, Asn, and Gin. Smallchelators chelates usually have 8 and 7 coordinating oxygen atoms forGd³⁺ and Ca²⁺, respectively.

For macromolecules such as proteins, the coordination numbers areslightly smaller possibly due to steric crowding effect. The averagevalues for Gd³⁺ and Ca²⁺ are 7.2 and 6.5, respectively. The pentagonalbipyramidal geometry and potential metal binding ligand residues wereused in creating metal binding site in CD2 with computer algorithm basedon parameters selected by the inventor. As Gd³⁺ is highly positivelycharged, more negatively charged residues were placed in thecoordination shell to increase the affinity for Gd³⁺ over Ca²⁺. Metalbinding sites with different numbers of coordination waters (q=1 and 2)have been designed in CD2 (denoted as CA.CD2). For example, CA1.CD2 (seebelow), the metal binding ligand residues are from different locationsof the protein to form a metal binding pocket (discontinuous) whileCA9.CD2 (see below) contains a the metal bind site formed by acontinuous EF-hand loop from calmodulin linked by a flexible glycinelinker. This site is designed to mimic previously reported highlyflexible contrast agent conjugated to macromolecule and to test ourhypothesis.

The amino acid sequences of exemplary contrast are shown in Sequence Id.Nos. 1 through 19.

The Computational Method for the Design of De Novo Binding Sites inProteins

The computation method can be used to designed metal binding sites intoscaffold proteins. For example, previously published research by theinventor described established parameters such as the pentagonalbipyramidal geometry of the most popular Ca²⁺ binding sites withpotential ligand residues such as the carboxyl groups of D, N, E, Qand/or mainchain carbonyl oxygen atoms. The resulting proteinpreferentially binds Ca²⁺ over Mg²⁺ (10 mM) and K⁺ (150 mM) atphysiological conditions.

These experiments resulted in metalloprotein designs that have a highcoordination number (seven) metal binding site constructed into aP-sheet protein. Gd³⁺ and Ca²⁺ share similar coordination geometry andproperties. To design de novo Gd³⁺ binding sites into proteins, theinventors started by carrying out detailed structural analysis of Gd³⁺binding sites in small chelators and proteins.

As shown in FIGS. 1A, 1B and 1C (CD2 derivative), the inventors havesolved the NMR structure for one of the designed metal binding proteins.Structural studies revealed that the coordination geometry in thedesigned proteins is the same as the designed structure. The structuralprofiles can assist in further designing of Gd³⁺ binding proteins. Gd³⁺binding proteins also can be designed using developed computationalmethods.

Gd³⁺ Binding Proteins can be Designed with Knowledge about Ca²⁺-BindingProteins

Gd³⁺ binding proteins can be designed using developed computationalmethods. Since Gd³⁺ has a high number of positive charges, more chargedresidues in the coordination shell increased the selectivity for Gd³⁺over Ca²⁺. To systematically evaluate the key factors for the metalselectivity, CD2 variants were created with different numbers of chargedligand residues (2 to 5) in the metal coordination shell. Metal-bindingsites with a higher number of negatively charged residues have strongeraffinities for Gd³⁺ and Tb³⁺ over calcium. The resulting proteinselectively binds Gd³⁺ over Ca²⁺ at physiological conditions with K_(d)values for Gd³⁺ and Tb³⁺<0.01 μM and for calcium >50 μM.

The inventors have designed Gd³⁺ binding sites in GFP in a pentagonalbipyramidal geometry with seven ligands using established computeralgorithms. Several of the designed sites at different locations in GFPare shown in FIG. 1C. GFP variants with designed metal binding sitesexhibit strong metal binding affinity for the Gd³⁺ analog Tb³⁺. Thisdemonstrates that a general method for designing metal binding sites hasbeen developed.

Affinity and Selectivity of Metal Binding

As Tb³⁺ has an ionic size similar to Gd³⁺, the developed Tb³⁺ sensitizedfluorescence resonance energy transfer (FRET) method may be used tomeasure the binding of Gd³⁺. See Ye Y., et al. Metal Binding Affinityand Structural Properties of an Isolated EF-Loop in a Scaffold Protein,Protein Eng, 14, 1001-13 (2001). This method can obtain the lower limitof metal binding affinities. The addition of Gd³⁺ competes for the Tb³⁺binding pocket and shows a decrease in Tb³⁺ FRET enhancement. Therefore,the Gd³⁺ binding affinity can be estimated. To accurately measure metalaffinity compared with the known stability constant of(ethylenedioxy)diethylene nitrilotetraacetate (EGTA), the inventors haveobtained the relative binding constant of EGTA for Tb³⁺ using the Tb³⁺FRET method under the same experimental conditions. Therefore, thestability of designed contrast agents can be accurately obtained.

Designed Contrast Agents Exhibit High MRI Proton Relaxivity

In the designing of preferred contrast agents, the following factorswere considered:

1) A high relaxivity, especially for T1, in order to obtain betterimages.

2) A specific in vivo distribution providing molecular imaging ofdesired targets.

3) The toxicity (negligible or low) of a metal-loaded contrast agent isa prerequisite as free Gd³⁺ is toxic. The contrast agent should bethermodynamically and kinetically stable, e.g. with high bindingaffinity and very low off-rate, which can minimize the release of freetoxic Gd³⁺. A contrast agent with high relaxivity and the capability totarget specific tissues or organs dramatically reduces its requiredconcentration for imaging and toxicity. In addition, a Gd³⁺-loadedcontrast agent does not compete with the proteins for metal binding.

4) The solubility and excretion time of the agents from the body toallow imaging and reduce toxicity. Directly coordinating Gd³⁺ ions byprotein ligand residues eliminates the high internal mobility of theparamagnetic moiety associated with polymers and protein conjugates.

Relaxation times, T1 and T2, were determined at 3 Tesla. T1 wasdetermined using inversion recovery and T2 using a multi-echoCarr-Purcell-Meiboom-Gill (CPMG) sequence. Samples were imaged andrelaxation parameters calculated prior to Gd³⁺-binding and after addingGd³⁺ in 1:1 molar ratio. As shown in Table 1 (below), the designedcontrast agent CA1.CD2—exhibits T1 relaxivity up to 48 mM⁻¹ s⁻¹, about a10-fold increase in the sensitivity compared to that of DTPA. Aconcentration of 50 μM, contrast agent CA1.CD2 is able to generate abright image while Gd-DTPA could not produce a visible image. Thissuggests that a significantly lower local concentration (˜10 μM) isenough for good imaging. In addition, several other designed CD2variants such as CA3.CD2-6D79 and EEDDN also exhibit relaxivityvalues >30 mM⁻¹ s⁻¹. On the other hand, CD2 with grafted EF-loop IIImodified from calmodulin (CA9.CD2) is similar in relaxivity to DTPA,possibly due to the intrinsic mobility of the metal binding site.

TABLE 1 T1 proton relaxivity of different classes of contrast agentsNumber Magnetic T₁relaxivity of field MW Type Compounds (mM⁻¹ s⁻¹) metalBo(T) (KDa) *Small GdDTPA 4.5 1 1.5 0.743 compound Designed ContrastAgents Sequence CA0.lac 4.2 1 3 14.5 ID No. 1 Sequence CA1.CD2** 48 1 312 ID. No. 2 Sequence CA1.CD2- 37 1 3 12 ID. No. 3 EEDDE SequenceCA1.CD2- 36 1 3 12 ID. No. 4 EEDDN Sequence CA1.CD2- 14 1 3 12 ID. No. 5EEDDQ Sequence CA1.CD2- 9 1 3 12 ID. No. 6 NENDN Sequence CA1.CD2- 34 13 12 ID. No. 7 EENDN Sequence CA2.CD2- 34 1 3 12 ID. No. 8 6D31 SequenceCA2.CD2- 37 1 3 12 ID. No.9 R31K Sequence CA3.CD2- 35 1 3 12 ID. No. 106D79 Sequence CA9.CD2 5 1 3 12 ID. No. 11 *From reference.

The proton relaxivities of designed proteins are not altered in thepresence of excess Ca²⁺ and a strong natural Ca²⁺ binding protein doesnot have an enhanced proton relaxivity. In order to assess the stabilityof Gd³⁺ binding under conditions mimicking the extracellular environmentand other biological metal ions on the relaxivities of the designed Gd³⁺binding proteins, the T1 parameter was measured in the presence of 1:1or 100:1 ratio of Ca²⁺ over Gd³⁺ (up to 10 mM Ca²⁺). The results inTable 1 show that the relaxivities of the instant developed contrastagents were not significantly affected in the presence of 100 foldexcess Ca²⁺.

The relaxivity of these designed contrast agents were further measuredwith tissues from different organs and serum of mice for more than twodays. No significant change of relaxivity values were observed, furthersuggesting those pure designed contrast agents have in vivo stability.

Samples were imaged and relaxation parameters calculated prior to andafter adding Gd³⁺ in 1:1 molar ratio. FIG. 3A shows the sample orderfrom top row left to right, then second row, left to right, then thirdrow left to right is: 1) dH2O, 2) Tris, 3) Gd-DTPA in H₂O, 0.10 mM Gd³⁺,4) Gd-DTPA in Tris, 0.10 mM Gd³⁺, 5) 0.092 mM Gd³⁺-w.t. CD2, 6) 0.077 mMGd³⁺-CA4.CD2, 7) 0.10 mM Gd³⁺-CA2.6D31, 8) 0.10 mM Gd³⁺-CA9.CD2, 9)0.020 mM Gd³⁺-CA1.CD2-GST, and 10) 0.050 mM Gd³⁺-CA1.CD2. FIG. 3B showsMR images produced using Spin-echo sequence, TR 6000 ms, TI 960 ms, TE7.6 ms at 3T. These results demonstrate success in creating Gd³⁺ bindingsites in scaffold proteins, which can preferentially bind Gd³⁺.

Correlation Time of Designed Metal Binding Proteins

The inventors also have applied high resolution NMR to probe the dynamicproperties of designed metal binding proteins. FIG. 4A shows the dynamicproperties of two engineered metal binding proteins. FIG. 4B shows theorder factors of CA1-CD2 with discontinuous ligand residues that havethe same dynamic properties as the scaffold protein. The T1 values ofCA9.CD2 with a continuous metal binding sites linked by a flexible Glylinker. The metal binding site has significantly higher flexibility thanthe scaffold protein due to the flexible linkers.

The overall correlation time of the designed metal binding protein is9.2 ns, consistent with proteins of similar size. The order factor S² ofthe ligand residues is similar to that of the average value of theprotein, suggesting that the metal binding pocket tumbles as a wholewith the protein. Therefore, the measured correlation time of theprotein directly reflects the TR of the metal binding site. On the otherhand, CD2 with the grafted EF-loop III of calmodulin appears to be moreflexible than the host protein. Taken together, these results show thatdesigned metal binding sites in proteins with minimized mobility canincrease proton relaxivity.

Serum Stability of Designed Contrast Agents

The serum stability of designed proteins were studied by incubating thedesigned proteins that complex with equal molar of GdCl₃ with humanserum. The relaxivities of exemplary contrast agents remained intact inthe presence of human serum and were not significantly affected in thepresence of 100 fold excess Ca²⁺. Further, these designed contrastagents remained intact after incubation in human serum for greater 48hours.

Developing Protein-Based Contrast Agents by Design

According to the theory developed by Blombergen, Solomon, and others andthese results, water q in the coordination shell, τ_(R) rotationalcorrelation time, and water exchange rates are the key factors of protonrelaxivity. An established design approach to designing additional Gd³⁺binding sites in domain 1 of CD2 and GFP can optimize the followingfactors to achieve even higher MRI relaxation signals.

1) Vary coordination geometry: Gd³⁺ is expected to have one or two morecoordinated oxygen ligands than Ca²⁺. It has been shown that designedmetal binding sites with pentagonal bipyramidal geometry and four tofive negatively charged ligand residues are able to bind Gd³⁺ stronglyand with good selectivity to Ca²⁺ and Mg²⁺. To test whether increasingligand atoms can increase metal binding affinity and selectivity, themetal binding sites in the scaffold proteins can be designed by varyingthe coordination geometries with a total of 6 to 8 ligand residues asshown in Table 2.

TABLE 2 Designed metal binding sites with different coordination numbersfrom proteins and water Total ligand numbers 1 H₂O 2 H₂O 3 H₂O 7 6 p 5 p4 p 8 7 p 6 p 5 p 9 8 p 7 p 6 p

2) Vary water number q in the coordination shell: Increasing the numberof water molecules in the coordination shell increases the relaxivitywhile the metal binding affinity and selectivity might be compromised.Metal binding sites with one to three hydrated water oxygen atoms in thecoordination shells with total coordination numbers of 7-9 have beendesigned (Table 3) to test if the maximum proton relaxivity can beachieved with two coordination water atoms without trading the metalbinding affinity and selectivity.

3) Vary ligand types and number of charged ligand residues: As Gd³⁺prefers more negatively charged ligands than Ca²⁺, a total of 2 to 6negatively charged ligand residues are designed by varying the number ofAsp or Glu as ligand residues as shown in FIG. 3 for site 7E15. Enrichednegative charges are expected to enhance the Gd³⁺-binding affinity andstability. It may also increase the relaxivity by facilitating theinner-outer water exchange.

4) Vary the locations of metal binding sites: A good choice of metalbinding site locations is essential not only for the protein folding andstability, but also for the proton relaxivity due to the effect ofprotein environment on the secondary and outer sphere water exchangeproperties. Table 3 lists the designed metal binding sites at differentlocations of CD2.

TABLE 3 Designed metal binding sites in CD2 Designed site Ligandresidues CD2-6D15 (CA4.CD2) N15D, N17D, N60, D62 CD2-7E15 (CA1.CD2)N15E, L58E, K64D, E56, D62 CD2-6D79 (CA3.CD2) A92E, T79D, N77, E33, N90CD2-6D31 (CA2.CD2) R31D, K43D, E29, E41

Several locations in both CD2 and GFP were selected based on thefollowing considerations (FIGS. 1A through 1E). First, these proteinvariants exhibit native like structure and folding properties. Inaddition, the fluorescence properties of GFP are not altered.Furthermore, the removal of the key residues involved in the binding toCD48 by mutation does not alter the structure of the proteins. The celladhesion function of CD2 is eliminated to allow CD2 to function solelyas a protein contrast agent. Second, all of these metal-binding sitesbind to Gd³⁺ analog Tb³⁺ as revealed by Tb³⁺ fluorescence energytransfer. Third, these metal binding sites have different secondarystructure and solvent accessibility, which allows testing whether thesecondary and outer sphere environment contribute to the waterrelaxation and metal binding affinity. As shown in FIGS. 1A, 1B and 1C,metal binding site (e.g. CA1.CD27E15) is formed by two residues in thebeta strand B and three ligand residues from the loop regions while allof the protein ligand residues of 6D79 are from the beta strands.

5) Vary the number of Gd³⁺ binding sites: As the quality of MR imagingis related to the relaxivity and concentration of the contrast agents,multiple sites will be engineered in a single polypeptide chain with theaim of increasing the local Gd³⁺ concentration without changing theprotein concentration. The designed Gd³⁺-binding sites usingnon-overlapped ligands can be tested individually first and thenengineered into a single protein. For example, it is possible to createa CD2 with all three Gd³⁺ binding sites of 7E15 (CA1.CD2), 6D79(CA2.CD2), and 6D31 (CA2.CD2). Similar approaches are used for GFP.Additional metal binding sites can be created by linking the tandemrepeats of CD2 and GFP with multiple metal binding sites. As shown inFIG. 1E, multifunctional contrast agents with high payload can becreated as CD2-GFP fusion proteins with multiple Gd³⁺ binding sites.

6) Vary the size of proteins: The size of the developed protein hasseveral effects on the properties of the contrast agents. According tothe present invention and as pointed out by Lauffer, the correlationtime to achieve optimal relaxivity under current clinical magnetic fieldstrength is 10-50 ns, which correlates to the protein with compactstructure of 10-30 KDa. Proteins with different domains might haveadditional internal motions between domains. In addition, thebiodistribution and circulation of contrast agents are also dependent onthe size of proteins. It is known that proteins with molecular weightsin the region of 10-60 KDa have been shown to be ideal for molecularrecognition and fast blood diffusion and excretion via the kidney.

Using the methods disclosed herein, the inventors have designed metalbinding sites in proteins such as domain 1 of CD2 (11 KDa), CD2-CD2tandem fusion proteins, GFP (28 KDa), CD2-GST fusion protein (38 KDa),CD2 and GFP fusion proteins (40 KDa) with different molecular weights.These designs allow the development of contrast agents with highrelaxivity optimized to the magnetic field strength, and biodistributionand bioelimination. In addition, Gd³⁺ binding sites embedded into stableproteins eliminate the high internal mobility of the paramagnetic moietyand optimal total correlation time and water exchange rates.Furthermore, these tandem repeats of CD2 and GFP containing multiplemetal binding sites achieve high payload agents with extremely highsensitivity and reduce the use of the amount of contrast agents andhence significantly reduce the toxicity is also significantly reduced.

Multi-Functionally Targeted Contrast Agents

Multi-functional contrast agents with both the capability for MR imagingand fluorescence optical imaging have been created. See, e.g. FIG. 1D.Fluorescence imaging can be utilized due to its characteristic highsensitivity. See, e.g., Sequence Id. Nos. 16 through 19. Proteins withboth paramagnetic binding sites and fluorescence properties have beengenerated by designing Gd³⁺ binding sites directly in GFP. Studies allowa direct comparison of the results from scintillography and PET,providing spatial information for the cause of the cancer and theprogress of the treatment.

Live Imaging

The distribution of Gd³⁺ in different organs of CD-1 mice was analyzedusing ICP-MS for Gd and immunological methods by employing antibodiesagainst the host protein CD2. Critical organs were collected andanalyzed. As shown in FIGS. 5A-D, which are chronological images, theimmunohistochemical staining studies further revealed that the designedprotein mainly localized to the cortex of the kidney, which isconsistent with a relatively strong MR image enhancement afteradministration of the prepared contrast agent. After injection of thecontrast agents the animal remained alive and exhibited normal behaviorfor >5 days. See. FIG. 5B and FIG. 5D, respectively. The organdistributions of the protein contrast agent were further verified byimmunohistochemical staining with kidney, liver, and lung tissues.

The in vivo T1 and T2 relaxivity values for Gd-CA1.CD2 in kidney are 49and 56.8 mM⁻¹ s⁻¹ kg tissue, which are significantly greater than thosereported values for Gd-DTPA in kidney (1.0 and 10.8 mM⁻¹ s⁻¹ kg tissue,respectively) These results are consistent with the in vitro relaxivitymeasured using purified protein. The in vivo R2 relaxivity in kidney isabout 11.6 fold greater than R1 relaxivity.

Toxicity

Briefly, testing cells (1×10⁴ cells/well in 100 μl medium) wereincubated with designed proteins with/without Gd³⁺ (up to 50 μM) for 48hours. The cell viability was analyzed by MTT assay. No toxicity wasobserved in any of the tested cells treated with an exemplary contrastagent (e.g. CA1.CD2) with concentrations up to 50 μM. For example, asshown in FIG. 6, no toxicity was observed in all tested cells treatedwith designed proteins with concentrations up to 50 μM. In addition, theeffects of contrast agent on liver enzymes (ALT, ALP, AST, LDH), ureanitrogen, bilirubin, and total protein from CD1 mice 48 hourspost-contrast injection was found to be negligible compared to a controlsubject. Furthermore, no acute toxicity was observed for mice aftercontrast agent injection, suggesting that the contrast agent of thisinvention are likely to maintain its metal complex stability and strongaffinity for Gd³⁺ in vivo.

The foregoing detailed description of the preferred embodiments and theappended figures have been presented only for illustrative anddescriptive purposes. They are not intended to be exhaustive and are notintended to limit the scope and spirit of the invention. The embodimentswere selected and described to best explain the principles of theinvention and its practical applications. One skilled in the art willrecognize that many variations can be made to the invention disclosed inthis specification without departing from the scope and spirit of theinvention.

1. A contrast agent comprising: a) a scaffold protein; and b) at leastone tailored metal ion chelating site, wherein the at least one metalion chelating site is integrated into the scaffold protein.
 2. Thecontrast agent as claimed in claim 1, wherein the at least one metal ionchelating site is embedded within the scaffold protein.
 3. The contrastagent as claimed in claim 1, wherein the at least one metal ionchelating site is substantially embedded within the scaffold protein. 4.The contrast agent as claimed in claim 1, wherein the reflexivity of thecontrast agent is greater than that of the scaffold protein.
 5. Thecontrast agent as claimed in claim 1, wherein the metal binding sitepreferentially binds an paramagnetic metal ion selected from the groupconsisting of Gd(III), Mn(II), Fe(II), Fe(III), Co(II), Co(III),Ni(III), Mo(V), and V(IV).
 6. The contrast agent as claimed in claim 1,wherein the metal binding site preferentially binds an ion of a metalselected from the group consisting of Lanthanide Series metals.
 7. Thecontrast agent as claimed in claim 1, wherein the scaffold protein is aprotein that naturally binds metal ion.
 8. The contrast agent as claimedin claim 1, wherein the scaffold protein is fluorescent protein having achromophore.
 9. A method for visualizing a cell, tissue, organism oranimal comprising the administration of the contrast agent of claim 1.10. The method as claimed in claimed 9, wherein the visualization is bynuclear magnetic resonance.
 11. The method as claimed in claimed 9,wherein the visualization is by optical assays.
 12. A method forpreparing a contrast agent comprising the steps of: a) selecting ascaffold protein b) constructing at least one metal ion chelating site;and c) operatively embedding the metal ion binding site into theprotein, whereby step (c) imparts contrast agent properties into thescaffold protein.
 13. The method as claimed in claim 12, wherein thecontrast agent has a greater reflexivity than the scaffold protein. 14.The method as claimed in claim 12, wherein the scaffold protein isselected according to: a) resistance to pH denaturation; b) resistanceto proteolytic cleavage; c) tolerance for mutations; and d) molecularsize.
 15. The method as claimed in claim 12, wherein the scaffoldprotein is selected to based on intrinsic fluorescence of the scaffoldprotein.
 16. The method as claimed in claim 12, wherein the at least onemetal binding site is constructed by: a) identifying an analyte bindingpeptide that binds a metal ion with specificity, wherein the analyte isa metal ion; b) ascertaining at least a portion of the amino acidsequence encoding the metal ion binding peptide; and c) tailoring theamino acid sequence encoding the analyte binding peptide into an metalbinding site.
 17. The method as claimed in claim 12, wherein thescaffold protein is selected from immunoglobulin proteins.
 18. Themethod as claimed in claim 12, wherein the scaffold protein is selectedfrom proteins derived from a fluorescent protein having a chromophore.19. A multiple functional probe comprising: a) a fluorescent proteinhaving optical properties; and b) at least one metal chelating siteoperatively integrated into the fluorescent protein, wherein the probeacts as a contrast agent in imaging applications.
 20. The probe asclaimed in claim 19, wherein the metal binding site preferentially bindsan paramagnetic metal ion selected from the group consisting of Gd(III),Mn(II), Fe(II), Fe(III), Co(II), Co(III), Ni(III), Mo(V), and V(IV). 21.The contrast agent as claimed in claim 19, wherein the metal chelatingsite preferentially binds an ion of a metal selected from the groupconsisting of Lanthanide Series.
 22. A method of generating enhancedimages of a subject comprising the steps of: a) administering a contrastagent having at least one metal ion chelating site operatively embeddedinto a scaffold protein; and b) generating one or more images of thesubject.
 23. The method as claimed in claim 22, wherein the one or moreimages are generated by nuclear magnetic resonance.